1. Field of the Invention
The present invention relates to a dry integral multilayer analytical element containing an oxidized coenzyme and a process for the preparation of said element.
2. Description of Prior Art
Reactions in which dehydrogenase and a coenzyme are involved have been widely employed in clinical chemical analyses. For example, various reaction systems in which a dehydrogenase such as glycerol dehydrogenase, cholesterol dehydrogenase, lactate dehydrogenase, alcohol dehydrogenase, glutamate dehydrogenase, aldehyde dehydrogenase, .alpha.-glycerophosphate dehydrogenase or glucose-6-phosphate dehydrogenase participates are employed for quantitative determination of substrate such as triglyceride, glycerol, cholesterol, lactic acid, glutamate, glycerol-3-phosphoric acid or glucose-6-phosphoric acid, and an enzyme such as aspartic acid aminotransferase (AST), lactate dehydrogenase (LDH), amylase or creatine kinase (CK). Through the reaction, quantitative analysis can be made by directly measuring increase or decrease of the amount of a reduced coenzyme. However, the conventionally employed NADH (i.e., nicotinamide adenine dinucleotide) or NADPH (i.e., nicotinamide adenine dinucleotide phosphate) has its maximum absorption peak at approx. 340 nm, and therefore the photometric measurement requires an expensive photometor for the measurement of a light in the ultraviolet region. Another drawback resides in that such measurement of a light in the ultraviolet region is easily influenced by a variety of coexisting compounds.
An alternative photometric analytical method using an electron acceptable dye-forming compound and an electron transmitter (i.e., carrier) in combination for forming a dye having an absorption peak in the visible ray region upon contact with the produced NADH was proposed for replacement of the above-described method of directly measuring the produced NADH (or NADPH). This method, however, has a problem that the reagent composition comprising the oxidized coenzyme, electron acceptable dye-forming compound and electron transmitter compound easily deteriorates to show lowering of reaction sensitivity when an analytical element containing such composition is stored for a long tiem of period. This problem is often observed particularly in the case that such composition is incorporated into a dry integrated multilayer analytical element as described in Japanese Patent Publication No. 53(1978)-21677 and Japanese Patent Provisional Publications No. 55(1980)-164356 and No. 60(1985)-222769.
Japanese Patent Provisional Publication No. 49(1974) -11395 proposes a multilayer analytical element in which the electron transporter compound among the above-mentioned three components is arranged in a different layer from the layer containing the remaining two components. Japanese Patent Provisional Publication No. 59(1984)-44658 discloses a method of dispersing a hydrophobic substance in a hydrophilic medium, whereby the electron transmitter does not substantially react with the dye-forming material. Further, Japanese Patent Provisional Publication No. 59(1984)-88096 proposes two methods, namely, a method of arranging the electron transporter compound and an electron acceptable dye precursor (i.e., electron acceptable dye-forming compound) in separate layers in the same manner as described in the above-mentioned Japanese Patent Provisional Publication No. 49(1974)-11395 and a method of arranging the two components in the same layer in the form of separate particles, so as that the two components do not substantially react with each other. However, in these cases (cases of separating the electron transporter component from the remaining two components), a reaction rate decreases because the progress of the reaction is predominantly defined by a diffusion speed of the separated reagents (particularly the electron transporter compound), and hence detection sensitivity is deteriorated.
It was found that when a composition for detecting the above-mentioned NADH (or NADPH) is used in a reagent layer of a dry integral multilaye analytical element (e.g., multilayer analytical elements for automatic chemical analysis as described in Japanese Patent Publication No. 53(1978)-21677, and Japanese Patent Provisional Publications No. 55(1980)-164356 and No. 60(1985)-222769), a dye-forming rate of the composition after storage at room temperature becomes less than 1/2 of the initial speed within several weeks. It has been confirmed that the decrease of the speed is mainly caused by the deterioration of NAD coenzyme (or NADP coenzyme) not by the deterioration of the electron acceptable compound which is referred to in Japanese Patent Provisional Publication No. 57(1982)-132061. For preventing such deterioration of the coenzyme in a buffer solution, a measure of incorporating a chelating agent and an azide in combination is described in Japanese Patent Provisional Publication No. 59(1984)-82398. However, the use of the azide having explosion characteristics and toxicity is unfavorable from the viewpoints of safety and environmental pollution.
It is known that the reagent layer containing NAD coenzyme or NADP coenzyme preferably has a pH value in the vicinity of neutral level in all stages including a coating stage in order to avoid the deterioration of coenzyme in the storage. Also known is that diaphorase loses its activity when it is placed under a pH condition of less than approx. 6.0. On the other hand, it is known that the reaction system preferably has an alkaline pH value when dehydrogenase participates in the reaction. However, there has not been proposed any dry analytical element element simultaneously satisfying the above-mentioned conflict of the appropriate pH conditions.